The thiazide-sensitive Na-Cl cotransporter (NCC) reabsorbs 5-10% of the filtered Na+ and Cl load in the kidney. Dysregulation of the NCC, either iatrogenic from diuretic use, or inherited, in Gitelman's syndrome, causes human disease. Gitelman's form of Bartter's syndrome results from mutations at any of several different sites within the transport protein; the Gitelman's phenotype is believed to result because mutations in the carboxy-terminal cytoplasmic domain of the NCC inactivates or strongly down-regulates its NaCl transporting capacity when expressed in Xenopus oocytes. Other preliminary experiments have identified three kidney proteins that bind to the NCC carboxy-terminal tail. The long term goal of these experiments is to delineate factors that regulate the thiazide-sensitive Na-Cl cotransporter, specifically the role of the carboxy-terminal domain of NCC. The first group of experiments in this proposal will examine the effects of mutations along the terminal 60 amino acids of the NCC on its capacity to transport Na+ and Cl, its binding affinity for Na+ and Cl, and its affinity for thiazide diuretics. For these studies, normal and mutated clones will be expressed in Xenopus oocytes. The mechanisms by which carboxy-terminal mutations reduce transport activity will be assessed by cloning a FLAG epitope into the protein and determining rates of protein synthesis and insertion into the plasma membrane. These experiments will test the hypothesis that carboxy-terminal mutations of NCC reduce its activity without altering NCC synthesis, protein processing, or insertion into the plasma membrane. In preliminary experiments the yeast two hybrid system was used to identify 3 proteins from a mouse kidney cDNA library that bind to the carboxy-terminal region of NCC. These proteins were identified as Erp60, aldolase, and a novel protein, thiabindin. One of these proteins, Erp60, was found to co-localize with NCC at the apical membrane of distal convoluted tubule cells. Preliminary data show that Erp60 can be precipitated from kidney cortical membranes with an anti-NCC antibody. Thus, the second group of experiments will investigate interactions between Erp60 and NCC. Regions of NCC that interact with Erp60 will be identified using a combinations of forward and reverse yeast two hybrid approaches. The effects of mutations that inhibit protein-protein interactions will then be assessed by expressing the mutated proteins in Xenopus oocytes. The underlying hypothesis of these experiments is that some Gitelman's mutations impair protein-protein interactions that are crucial for maintenance of transport activity.